DE10105208A1 - Enzymatic production of cDNA by reverse transcription, in the presence of betaine, provides a high yield of full-length molecules from large RNA templates - Google Patents

Abstract

Enzymatic preparation (M1) of cDNA from RNA at least 3 kb long, by reverse transcription in a medium that includes betaine (I), is new. An independent claim is also included for use of (I) to loosen the secondary structure of RNA matrices.

Description

The present invention relates to a method for manufacturing of cDNA using reverse transcription of long chain RNA a length of at least 3 kb, at which the yield of full permanent cDNAs compared to conventional methods is increased.

Genetic information lies in the form of chains in the cell shaped macromolecules built up from nucleic acids are. The latter each contain a sugar portion (ribose or Deoxyribose), one of the bases adenine, guanine, or cytosine Thymidine or uracil, as well as phosphoric acid. The order of Bases within the nucleic acid chains form the basis for the genetic code. Because of this importance of the bases, the individual nucleic acids are also simply referred to as "bases" and the length of the nucleic acid chains based on the number of bases give. 1000 bases are referred to as "1 kb".

Deoxyribose-containing nucleic acid chains (DNA) form the permanent information store at the core of the cell while Ribose-containing nucleic acid chains (RNA) when in the Form of messenger RNA (mRNA) are present, the transfer of the in  the DNA stored information at the place of manufacture each encoded proteins (amino acid sequences) in the cell serve. Other RNAs such as ribosomal RNA are used not as information carriers, but are in the process of building up cellular structures such as ribosomes involved.

DNA is usually in the cell as a double strand of two opposite complementary single strands, the Double strand formation through hydrogen bonding between the bases adenine and thymine or guanine and cytosine. In contrast, RNA is only single-stranded.

The DNA is structured in such a way that the individual Protein-coding information is arranged in genes, where three bases encode an amino acid. If a pro produced in the cells of a certain tissue (expri is to be lubricated), the nucleus of the cell is first one Copy of the corresponding gene made in the form of an mRNA. This mRNA can leave the cell nucleus and join the manufacturing process place in the cell. The base sequence of the mRNA corresponds the base sequence of the respective gene coding for the protein, where the base of the thymidine of the DNA in the mRNA is the base Uracil kicks. So if you know the base sequence of an mRNA, let based on the genetic code Structure, d. H. the amino acid chain of the respective mRNA determine the encoded protein.

Because of this, the study is in certain cells or a particular tissue type mRNA present to one of the important most important fields of work in the field of modern Become molecular biology. Next to it is the base sequence other RNAs of interest as an investigation of these RNAs based cell structures have knowledge of this base sequence presupposes. While for the analysis of DNA a variety of Methods available are direct analysis of RNA complicated by the fact that RNA is unstable and not easily through Insertion in vectors and subsequent transfer in, for example  Bacterial cells (cloning) increased and analyzed can be.

To some extent, these problems could be solved by anyone after it had appeared - around 1970 - that some viruses, called retroviruses, have an enzyme have, which is able, starting from an RNA as Ma trize to produce a DNA, which is a true copy of the RNA is, with the exception that again in place of the uracil a thymidine occurs. Such, by "enzymatic copy DNA made from RNA is called "copy-DNA" or "cDNA" referred to, while the corresponding enzymes as "reverse Transcriptases "and the copying reaction they performed be called "reverse transcription".

For the purposes of the present invention, “reverse Transcription "thus refers to a process in which, starting from an RNA using reverse transcriptase becomes. This cDNA can be cloned in the same way analyze like natural DNA. In particular, can from the bases follow the cDNA to the base order of the underlying RNA and thus, in the case of mRNA, on the amino acid sequence of that of the mRNA-encoded protein can be closed.

The reverse transcription of RNA to obtain the correspon cDNA takes place in reaction approaches that are essential Ingredients at least a sufficient amount of reverser Transcriptase in a medium suitable for transcription, a mixture of the four required for the formation of the DNA Deoxyribonucleic acids and the RNA serving as a template These reaction batches are usually liquid. Possible is also that individual components of the reaction batch, for example the reverse transcriptase or the one to be copied RNA, bound to a solid matrix that is in contact with the liquid mixture of the other ingredients.  

The exact composition of such reaction approaches depends inter alia from the reverse transcriptases used and is basically known to the person skilled in the art [Sambrook et al., Mole cular Cloning - A Laboratory Manual, Cold Spring Harbor Labora tory Press, (1988)]. Furthermore, information about the exact Amount of components to be used as well as information on Carrying out the reverse transcription also the indications of the Manufacturer of the reverse transcriptases (e.g. Roche Diagnostics, Penzberg, Germany).

However, none is common with reverse transcription Obtain complete cDNA because the reaction stops beforehand. This can be due to the fact that the mRNA serving as a template contains longer complementary sections that correspond by appropriate formation of hydrogen bonds between adenine and Thymine or guanine and cytosine to form internal dop pel strands, d. H. lead to the formation of loops. The on this Secondary structures arising in this way make it the reverse Transcriptase often fails to produce a complete cDNA places [Abbotts, J. et al. (1993) "Mechanism of HIV-1 reverse transcriptase. Termination of processive synthesis on a natural DNA template is influenced by the sequence of the template-pri mer stem. "J Biol Chem 268, 10312-23; Brooks, E.M. et al. (1995) "Secondary structure in the 3 'UTR of EGF and the choice of reverse transcriptases affect the detection of message diver sity by RT-PCR "Biotechniques 19, 806-12, 814-5; Kuo, K.W. et al. (1997) "Intrinsic secondary structure of human TNFR-I mRNA influences the determination of gene expression by RT-PCR. "Mol Cell Biochem 177, 1-6]. It has been shown that in particular long chain RNAs, d. H. RNA with a length of at least 3 kb tend to form such secondary structures.

Prerequisite for the successful implementation of many moleku larbiological examinations and procedures is the Availability of complete cDNA, since only by means of complete cDNA also the entire base sequence of the RNA can be determined. This applies in particular to the production of so-called cDNA libraries  or when studying the edge sequences of RNA using PCR (polymerase chain reaction). In recent years, centered efforts to increase efficiency of cDNA production primarily to optimize the right verse transcriptases.

In addition, attempts have been made to improve the efficiency of reverse trans increase by adding substances that increase the Detach secondary structures from RNA. To that in this context investigated substances include, for example, trehalose, a substance originally from E. coli that is found in these Bacteria is synthesized in response to stress. His main function is to protect proteins from thermal denatu protect by the movements within the back proteins are minimized [Butler, S.L., and Falke, J. J. (1996) "Effects of protein stabilizing agents on thermal backbone motions: a disulfide trapping study. "Biochemistry 35, 10595-600; Singer, M.A., and Lindquist, S. (1998) "Multiple effects of trehalose on protein folding in vitro and in vivo. " Mol Cell 1, 639-48]. The molecule also has the same effect for RNA. It is therefore already in reverse transcription have been used [Carninci, P. et al. (1998) "Thermostabiliza tion and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA. "Proc Natl Acad Sci USA 95, 520-4].

Attempts have also been made to use betaine (trimethylglycine; Boncompa gni, E. et al. (1999) "Occurrence of choline and glycine betaine uptake and metabolism in the family rhizobiaceae and their roles in osmoprotection. "Appl Environ Microbiol 65, 2072-7) to Stei to reduce the yield of cDNA. With the so far However, studies have shown no effect on betaine (Vale ntin et al., (2000) European Journal Biochemistry, 267, 5438).

The use of trehalose, as well as other substances, leads to an increase in the production yield of long chain cDNAs. However, in the state of the  Technically known methods still not a satisfactory step reduction in yield.

It is therefore an object of the present invention to provide a method provide by means of which the yield of complete cDNAs in reverse transcription of long-chain RNAs the prior art is increased.

According to the invention, the object is achieved by the method according to An spell 1 solved.

The invention thus relates to a method for enzymatic Production of cDNA by means of reverse transcription of RNA with a length of at least 3 kb in a reverse transcriptase containing reaction mixture, which is characterized that betaine is added to the reaction mixture.

In the context of the present invention, it has turned out to be more surprising demonstrated that adding betaine to the reaction sentence for the reverse transcription of RNA with a length of at least 3 kb a higher yield of complete cDNA is aimed. This is in contrast to the results of Va lentin et al. (see above), which have no effect of betaine on the ef efficiency of reverse transcription (see Valentin et al., P. 5442, right column, third paragraph). al However, the authors only examined reverse there Transcription of a short (approx. 700 base long) mRNA. the compared to the present invention relates to the use of Be tain for long-chain, d. H. at least 3 kb long RNAs.

Surprisingly, the effects of betaine are greater the more longer is the RNA serving as a template. This is what makes it up Process according to the invention is particularly valuable since the problem of incomplete cDNAs primarily in long-chain RNAs occurs (see above).  

According to the invention, the RNA used has a length of min at least 3 kb. According to the invention, the RNA can either be in a mixture with other, smaller and / or larger RNAs are available as well as already in a purified form (for Purification of RNAs see Sambrook et al., 1988 (see above))

According to a preferred embodiment, the one used RNA at least 4 kb in length, according to one particularly before preferred embodiment of at least 7.5 kb.

According to a particularly preferred embodiment, that is too RNA copying an mRNA, being included according to the invention is that the mRNA to be copied is either in a mixture with other RNAs, or that they already exist from the other RNAs according to cleaning steps known to those skilled in the art [Sambrook et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)].

The concentration of betaine in the reaction mixture can, for example example, be from 0.1 to 3 M, preferably from 0.5 to 2.5 particularly preferably from 1 to 2 M, and is very particularly present increases at about 2 sts, this information, as well as all following Concentration information, on the total volume of the reaction mixture zes are related unless otherwise stated.

The method according to the invention can be used with any reverse trans be performed. In particular, this is fiction appropriate procedures, with AMV and M-MuLV (Moloney Murine Leukemia Virus) reverse transcriptases to be performed.

According to a preferred embodiment of the method, one adds additionally trehalose to the reaction mixture.

The addition of trehalose has the surprising effect that the Efficiency of cDNA production well beyond measure that is achieved with betaine or trehalose alone  becomes. This leads to an unforeseeable synergistis chan effect of betaine and trehalose (see example 2).

Trehalose can, for example, in amounts of 0.1 to 1.5 M. Reaction approach can be added. According to a preferred Embodiment is the concentration of trehalose in the reak tion approach from 0.3 to 1 M, and most preferably about 0.6 M.

In the context of the invention, it has surprisingly been found that Betaine the secondary structure of RNAs with the length of at least can solve at least 3 kb. The invention therefore also relates to the use of betaine in the dissolution of secondary structures ren of RNA, the length of the RNA is preferably at least 3 kb at least 4 kb, and most preferably at least 7.5 kb is.

The invention is illustrated below by figures and examples explained.

Brief description of the figures Fig. 1

• A) [α ³² P] dCTP-labeled cDNA from different reverse transcription reactions, which were carried out under different conditions, separated by electrophoresis on a denaturing agarose gel and analyzed by means of a phosphorimager.
  Lane a: control (no betaine, no trehalose)
  Lane b: 2 M betaine
  Lane c: 0.6 M trehalose
  Lane d: 2 M betaine and 0.6 M trehalose
  The molecular weight data correspond to the marker bands (1 kb ladder), the white arrows indicate regions in which the cDNA synthesis was terminated, this problem appearing to be solved under the various experimental conditions.
• B) Densitometric representation of the autoradiograph of panel A.
  The migration route was plotted against the RFU value (Relative Fluorescence Unit) from the phosphorimager analysis.
  Drawing 1: control (no betaine, no trehalose)
  Drawing 2: 2 M betaine
  Drawing 3: 0.6 M trehalose
  Drawing 4: 2 M betaine and 0.6 M trehalose
  Vertical solid and dotted lines represent 3 kb and 7.5 kb molecular weight, respectively.
• C) [α ³² P] dCTP labeled cDNA from reverse transcriptions, which were carried out with increasing betaine concentrations. The cDNAs were separated by means of denaturing agarose gel electrophoresis and exposed to a phosphoimager screen.

Fig. 2

Schematic representation of the PCR leading to the evolution of Effi ciency of the first strand synthesis was carried out, the First strand synthesis with specific primers for different Regions of mouse chlathrin mRNA was performed.

The diagrams show the number of PCR cycles that had to be carried out until the crossing point (see below) was reached.
C: control
B: 2 M betaine
T: 0.6 M trehalose
B + T: 2 M betaine and 0.6 M trehalose

Fig. 3

Secondary structure of the bovine oxytocin receptor mRNA fragment, calculated by the sugar-RNA convolution log and darge provided by RNA-draw-V 1.1.

Fig. 4

Melting curves of

• a) complex mRNA from mouse muscle tissue
• b) 297 base fragment of the bovine oxytocin receptor

Solid line: control.
Dotted line: 2 M betaine

Examples example 1 Reverse transcription

After priming with 500 ng oligo-dT ₂₅ in a total volume of 30 μl, 5 μg total RNA were subjected to a reverse transcription, 10x first strand buffer (500 mM Tris-HCl, 750 mM KCL, 15 mM MgCl ₂ , pH 8.3 at 20 ° C), 10 mM DTT, 500 uM dNTPs, 200 U MMLV reverse transcriptase (Gibco BRL, ML) were used. The reverse transcription was carried out according to the manufacturer's protocol at 42 ° C.

For more efficient first strand synthesis either 1-3 M betaine (made from a 5 M stock solution), 0.6 M trehalose (made from a 2 M stock solution) or a combination of these two compounds was added. The following temperature profiles were used:
Adding betaine: 90 minutes at 42 ° C
Add 0.6 M trehalose: 30 minutes at 42 ° C, then one hour at 60 ° C
Add 2 M betaine and 0.6 M trehalose: 30 minutes at 42 ° C, then one hour at 60 ° C

A 5 µl aliquot was reverse transcribed in the presence of 0.2 µl (α ³² -P) dCTP (100 µCi per µl, Amersham). All experiments were carried out in triplicate.

Example 2 Denaturing gel electrophoresis

The labeled samples from the reverse transcription were over Night in a 1% agarose gel in 30 mM NAOH, 1 mM EDTA at 1 volt / cm subjected to electrophoresis. The gel was on a nylon membrane (Nytran, Schleicher and Schuell) dried, to avoid losses during the drying process. The Membrane / gel sandwich was a phosphor imager screen 16 hours long exposed. The result was measured using a Storm ™ Phosphoimager (Molecular Dynamics), with a resolution of 50 µm pictured, and the total amount of signals as well how the distribution of the signals was measured using the ImageQuant ™ Software analyzed.

Fig. 1A shows the autoradiographs as the results of the experiments carried out under various conditions. Two major effects can be observed. The most striking is the increase in the average size of the synthesized cDNA in the presence of betaine alone (lane 2) and trehalose alone (lane 3). The second effect is the elimination of various strong bands that occur alone in the control lane and in the lane with trehalose, shown by white arrows. These bands appear to be the product of breaks in cDNA synthesis, probably due to secondary structures present in some highly expressed mRNAs. In the lanes with cDNA, which were produced in the presence of betaine (lanes 2 and 49, these bands decrease significantly. In general, it can be said that these lanes appear generally more homogeneous.

FIG. 1B shows the quantification of the signal distribution in different size windows ver of the synthesized cDNA. More specifically includes 1B densitometric plot showing the distribution of the built-in "signals", as the RFU. Show (Relative Fluorescence Units relative fluorescence units), which he were averages by phosphorimager. Also shown are two lines which show different molecular sizes of the cDNA (solid line 3 kb; dotted line 7.5 kb). The area under the curve was quantified and the ratio between the respective RFU and the control was calculated and is shown in Table 1.

Table 1

Densitometric analysis of the built-in radioactive label in different size windows of the cDNA

Fig. 1C shows that the optimal betaine concentration is 2 M be.

Different conclusions can be drawn from this data The inclusion of betaine under trehalose in the reverse Transcriptional mix leads to an increase in the synthesis of cDNA with a higher molecular weight. In the area below below 3 kb trehalose outperforms betaine. In the middle area  between 3 kb and 7.5 kb show both betaine and trehalose an effect whereby trehalose appears more efficient. Erstaunli Betain and Treha are usually in the range above 7.5 kb loose equally efficient. A combination of both connections led to a five times higher installation of a radioactive mar kierung. Therefore, a cooperative effect of the two appears Connections likely, leading to a significant increase of products with long chains.

Example 3 Real time quantitative PCR

The aim of these experiments was to investigate whether through a Add betaine and trehalose and a combination of the two Connections to reverse transcription reaction approach actually Lich larger amounts of longer cDNA can be produced. Various Pri mer pairs (see Table 2) selected, each fragments result that differed at positions in the respective cDNA far from the oligo dT priming site.  

Table 2

In these experiments, the crossing point (number of Cycles that randomize to fluorescence above 0.2 Units) of quantitative real-time PCR as more suitable Marker used to count the number of trans under reverse to examine the resulting cDNA products.

The experiments were carried out as follows:
2 µl of the reverse transcription reactions carried out in the triplicate were diluted 20-fold and 2 µl were used as a template of the real-time qualitative PCR. The PCR was carried out in a LightCycler ™ instrument (Roche, Basel) using the FastStart Kit (Roche Diagnostics). The primer pairs shown in Table 2 were used. The distances of the clathrin primers from the oligo-dT priming site were 2435 base pairs (clathrin 1), 4006 base pairs (clathrin 2) and 12053 base pairs (clathrin 3).

The appearance of the resulting PCR products was determined by Elek Confirm trophoresis on a 1.3% agarose gel in TBE buffers Untitled. The crossing points of the real-time fluorescence curve were calculated using the accompanying data analysis software.

Fig. 2 shows that cDNA with a length of 3 or 4.5 kb, starting from the oligo-dT priming site, was synthesized in the same amount. At a distance of 12.5 kb, however, there is a significant increase in the amount of cDNA synthesis products if betaine or trehalose was added to the reaction mixture. There is also a significant additive effect when both compounds are used together. The average number of cycles to the cross point are 31.5 +/- 0.7 for the control and 27.7 +/- 3.8 for betaine and trehalose in combination. If one assumes a PCR efficiency of 1.74 (calculated from the increase in the linear amplification phase), this means that a combination of betaine and trehalose produces about 8.5 times more long-chain transcription products. The same was found in similar experiments when the starting point for the reverse transcription was not clathrin but β-actin (data not shown).

Example 4 Effects of betaine on the secondary structure of mRNA

With mRNA, the occurrence of secondary structures is a high organ nized property of the mRNA, which is determined by the environmental conditions gene (e.g. ionic strength) and influenced by the sequence of the mRNA becomes. The transition between the ordered and the disordered Stage is accompanied by an interruption of the base pair binding within the RNA. This leads to a hyperchromicity effect as a consequence of the separation of overlapping P-electron systems of heterocyclic bases (Michelson, A.M. (1963). The Chemi stry of Nucleosides and Nucleotides (N.Y .: Academic Press), pp. 444), and can be easily monitored by UV absorption changes with rising temperatures are monitored.  

To the possible dissolving effect of betaine on the secondary To investigate the structure of mRNA, we performed melting curves tests in the presence of 2 M betaine. We used mRNA from mouse muscle tissue and a 297 transcribed in vitro Base cRNA fragment of the bovine oxytoxin receptor transcript as Example of a complex mRNA pool and for a single one mRNA species. The latter was due to its high GC content (74%) and the highly stable and complex predicted seconds structure selected with a free energy of -349.32 kJ. The bovine oxytoxin receptor transcript also shows several Regions with a self complementarity of 88 nucleotides (from which are 55 GC pairs).

To be more precise, the RNA melt experiments were carried out as follows:
The RNA melting curve experiments were carried out in a closed, Peltier-heated cuvette system (Gilford, 2600 photometer), 12 μg mouse muscle mRNA, which had been purified three times with Oligotex ™ latex beads (Quiagen, Germany), or 12 μg des 297 base cRNA fragment of the bovine oxytoxin receptor transcript was used, which had been synthesized by in vitro transcription and had been purified on a column. The melting curve experiments were carried out in 0.5 N NaCl / 1 × PBS or 0.5 M NaCl / 1 × PBS / 2M-betaine. A melting curve with 0.5 M NaCl / 1x PBS / 2M betaine without RNA was used as a control and subtracted from the RNA-containing samples.

The cuvette was heated at 1 ° C per minute, data points were collected at 0.5 ° C. The T _{m of} the melting curves was calculated by a non-linear fitting of the data points (sigmoidal Boltzmann approximation). To be sure that the resulting hyperchromicity was not due to RNA degradation, the samples were cooled to room temperature for 6 hours after the experiment and then measured again. The optical density of the samples after cooling was within the 3% variance of the original absorption values. All melting curve experiments were carried out in triplicate.

Figure 4 shows the two melting curves resulting from the experiments, which were carried out either in the absence (solid line) or in the presence of betaine (dotted line). The result is that betaine is actually able to resolve secondary structures of RNA. In the case of the complex mRNA pool ( FIG. 4a), an increase in hyperchromicity can be seen at practically all temperatures compared to the control. The calculated T _m is 72.58 ° C +/- 0.083 ° C for the control and 67.07 ° C +/- 0.72 ° C for 2 M betaine. This increases the total hyperchromicity from 16.6% +/- 0.8% to 18.6% +/- 0.6% at 96 ° C. The same results are obtained in the case of the oxytoxin receptor cRNA fragment ( FIG. 4b). An increase in hyperchromicity can be seen, the calculated T _m is 86.57 ° C +/- 2.3 ° C and 78.93 ° C +/- 2.8 ° C for each control and 2 M betaine. The total hyperchromicity increases from 20.9% +/- 1.2% to 25.6% +/- 2.1%.

Both curves (both for the complex mRNA and for the individual cRNA) give an image with many phases. Everyone about course within the curves can be interpreted as that at this temperature under the given conditions Part of the molecule melts. Because RNA structures from different regions that consist of duplexes, bulges, loops or one cell region, leads to the melting of these regions to transitions that appear as independent peaks when the first derivation of the extension versus temperature will. In the case of the oxytoxin receptor mRNA fragment the predicted bases contribute to the multiphasic curve. In the case of complex mRNA with a significantly reduced Transitional picture, it seems to be some common property that distinguish all mRNAs (i.e. poly (A) tails) for the transition picture are responsible or that the transitions one The result of various secondary structures is that for some  few highly expressed mRNAs are typical in muscle tissue (i.e. GAPDH, β-actin).

Example 5 Calculation of secondary RNA structures

The secondary structure of the RNA fragment transcribed in vitro of the bovine oxytoxin receptor was determined by the sugar algorithm (Jaeger et al., (1980) Improved predictions of secondary struc tures for RNA. Proc Natl Acad Sci USA 86, 7706-10) calculated, and displayed using the RNAdraw V1.1 software (Matzura, O., and Wennborg, A. (1996). RNAdraw: an integrated program for RNA secondary structure calculation and analysis under 32-bit Microsoft Windows. Comput Appl Biosci 12, 247-9). A tempera The temperature of 42 ° C served as the basis for the calculation, since this is the optimal one Temperature for reverse transcription with MMLV (Murine Molo ney leukemia virus) is reverse transcriptase.  

SEQUENCE LISTING

Claims (12)

1. Process for the enzymatic production of cDNA by reverse transcription of RNA with a length of at least 3 kb in a reverse transcriptase-containing reaction mixture, characterized in that betaine is added to the reaction mixture. 2. The method according to claim 1, characterized in that the Concentration of betaine in the reaction mixture from 0.1 M to 3 M is. 3. The method according to claim 1 and 2, characterized in that the concentration of betaine is about 2M. 4. The method according to claims 1 to 3, characterized in net that the RNA has a length of at least 4 kb. 5. The method according to claims 1 to 4, characterized in net that the RNA has a length of at least 7.5 kb. 6. The method according to claims 1 to 5, characterized in net that the RNA is a messenger RNA (mRNA). 7. The method according to claims 1 to 6, characterized in net that you add trehalose to the reaction mixture added. 8. The method according to claim 7, characterized in that the Concentration of trehalose is from 0.1 M to 1.5 M. 9. The method according to claims 7 or 8, characterized net that the concentration of trehalose is about 0.6 M. 10. Use of betaine in the dissolution of secondary structures ren of RNA, the length of the RNA be at least 3 kb wearing.   11. Use according to claim 10, characterized in that the RNA has a length of at least 4 kb. 12. Use according to claims 10 or 11, characterized records that the RNA is at least 7.5 kb in length has.